Caveats in the study of perinatal behavioral development: utility of fetal study.

Study of behavior during development presents psychobiologists and neurobiologists with a unique set of problems that should be addressed in the design and analysis of experiments. Some of these caveats have become apparent only with the recent growth in research with subjects around the time of birth. For example, physiological regulation within the maternal-infant dyad, litter effects, the influence of context at the time of testing on performance, and dissociation of age-related change and experience, all are important experimental considerations in developmental study. Manipulation and measurement of behavioral variables in the fetus in vivo can provide one means for circumventing many of the methodological pitfalls that are associated with behavioral study of newborn subjects.

Prenatal experience with milk: fetal behavior and endogenous opioid systems.

The existence of organized responses to milk in newborn mammals, which lack experience at the nipple, implies the prenatal development of neural and behavioral systems for recognizing, obtaining, and processing milk. Many components of milk-directed behavior have been identified in the fetus. The stretch response expressed by neonatal rats during milk ejection at the nipple can be elicited before birth by infusing milk into the mouth of the fetus. Milk promotes reorganization of fetal motor behavior, facilitates expression of the stretch response, and alters fetal responsiveness to cutaneous stimulation. Pretreatment of fetuses with opioid agonists and antagonists has confirmed involvement of the mu and kappa opioid systems in mediating the effects of milk. Opioids appear to play a dual role in milk-oriented behavior: Initially, opioids suppress behavioral responses of the fetus and neonate to novelty, permitting ingestion of milk, and secondarily, opioid activity can promote learning at the nipple by functioning as a reinforcer. Study of milk-directed behavior in the fetus may promote better understanding of the special needs of preterm human infants.

Dynamical entropy is conserved during cocaine-induced changes in fetal rat motor patterns.

Our previous studies demonstrated that the intra-cisternal (IC) administration of cocaine to fetal rats increased motor activity and decreased responsiveness to perioral stimulation. One explanation for these observations comes from the behavioral pharmacology of stimulant drugs: increased motor activity is often associated with a decrease in its variety. Previous power spectral transformation of this data suggests an alternative explanation: cocaine-induced hyperactivity fixates a new behavioral pattern with complexity equal to that of saline controls. We explore these possibilities using statistical techniques derived from studies of nonlinear dynamical systems, examining patterns of the total motor activity of the individual fetus as counts per 5 s interval on either gestational day E20 or E21 for 20 min following IC injections of saline, 2.5 or 10 mg/kg of cocaine. The results are consistent with a state in which increased spontaneous activity is associated with the emergence of a new dynamical pattern which conserves entropy and provides experimental support for a fundamental conservation-variational relation, hT approximately equal to lambda 1 x DR, that has been proven for abstract models of chaotic dynamical systems. A multivariate analysis of variance (MANOVA) followed by appropriate analyses of variance (ANOVAs) and pairwise comparisons revealed that, whereas cocaine induced increases in the total amount of motor activity, the rate of increase in the variety of new sequences in activity counts over time did not change with treatment and age conditions. This invariant is quantified by an absence of change in topological entropy, delta hT = 0. The analyses also showed that, in order to maintain hT values, compensatory changes took place in the leading Lyapounov characteristic exponent, lambda 1 (the distance between sequential values 'stretched' along the increasing amplitudes of the variations) such that delta lambda 1 > 0, and the correlation dimension, DR (the hierarchical range of possible values, 'complicated clustering') was reduced, so that delta DR < 0. Our findings are consistent with the idea that the association between cocaine-induced increases in activity and decreases in adaptive response are not due to the dynamical entropy loss of decreased behavioral variety, but are rather the result of competitive interference by a drug-induced, equally complex, new pattern of spontaneous behavior.

Classical conditioning of opioid activity in the fetal rat.

Milk promotes activity in the kappa opioid system of the rat fetus that reduces responsiveness to cutaneous stimulation. In this study, fetuses on Gestational Day 20 were presented with an artificial nipple (conditioned stimulus; CS) paired with an intraoral infusion of milk (unconditioned stimulus; US). One paired presentation of the CS and US reduced fetal responsiveness after reexposure to the CS. Selective antagonism of opioid receptors after conditioning indicated that reduced responsiveness was due to mu opioid activity. Mu and kappa opioid activity was evident after 3 paired presentations of CS and US and reexposure to milk. Kappa opioid activity during conditioning was necessary for mu involvement after reexposure to the CS or US. These experiments, which were conducted with fetal subjects that lacked suckling experience, suggest that neurochemical systems engaged during suckling may change rapidly after the newborn's initial experiences at the nipple.

Behavioral effects of milk in the rat fetus.

Intraoral infusion of milk to the rat fetus promoted changes in behavior (mouth and rearlimb movements), reduced responsiveness to perioral cutaneous stimulation, and resulted in expression of a fetal stretch response. Milk also altered the temporal organization of fetal movements over periods up to 30 min. The orosensory characteristics of milk, in the absence of ingestion, was sufficient to evoke these behavioral effects. Reduced responsiveness to a perioral stimulus had a rapid onset (< 30 s) and brief duration (< 5 min). The ability to disrupt changes in motor organization and the stretch response with an intervening stimulus also exhibited a time course after milk infusion. The findings that tactile stimulation and biomechanical manipulations of fetal movement promote rearlimb activity and facilitate expression of the stretch are consistent with a dynamic perspective of early motor development in the fetus.

Opioid control of the fetal stretch response: implications for the first suckling episode.

Upon their first experience with milk, fetal rats express a stretch response that is similar to the postnatal behavior exhibited by infant rats at the nipple. Fetuses also possess a functional opioid system that is activated by prenatal milk exposure. The opioid receptor antagonist naloxone and the specific kappa antagonist nor-binaltorphimine blocked the stretch response and prevented the increase in rearlimb activity that is typically induced by milk. The mu antagonist beta-funaltrexamine blocked the stretch while permitting the expression of rearlimb activity. The kappa agonist U50,488 promoted rearlimb activity in the absence of milk, whereas the mu agonist [D-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) exerted little influence on fetal behavior. Fetuses pretreated with U50,488 stretched to nonmilk stimuli (saline or lemon), but fetuses pretreated with DAMGO did not. Opioid activation is part of a chain of events that culminates in the fetal stretch response and may be important in promoting milk ingestion during the newborn's first suckling episode.

Habituation to chemosensory stimuli in the rat fetus: effects of endogenous kappa opioid activity.

On Day 21 of gestation, rat fetuses respond to chemosensory stimuli by expressing stereotypic facial wiping behavior. A series of 4 experiments was conducted to investigate (a) the influence of morphine on fetal responsiveness to a single chemosensory infusion, (b) the effect of naloxone blockade of endogenous opioid activity on diminished fetal responsiveness over a series of chemosensory infusions, (c) the effect of endogenous opioids on the recovery of fetal responsiveness to infusion after various dishabituation procedures, and (d) the influence of selective mu and kappa opioid receptor antagonists on fetal habituation. These experiments confirm that fetuses habituate after a brief series of chemosensory infusions and that dishabituation promoted by presentation of a novel stimulus is facilitated by pharmacological blockade of kappa opioid receptors. Endogenous activity in the kappa opioid system may be functional in modulating the sensory environment around the time of birth.

Kappa opioid mediation of fetal responses to milk.

A series of experiments provided evidence for the existence of a functional opioid system in the fetal rat near term. Application of a tactile probe to the perioral region of the fetus consistently evoked a stereotypic facial wiping response. Administration of low dosages of morphine to the fetus had little effect on nonevoked motor activity but reduced fetal responsiveness to cutaneous stimulation. Milk infused into the mouth of the fetus reduced fetal responsiveness to the tactile probe. Milk's effect on cutaneous responsiveness was reversed by injection of the nonspecific opioid antagonist naloxone. The effect of milk on fetal responsiveness to cutaneous stimulation was reversed by the kappa opioid antagonist nor-binaltorphimine, but not by the mu antagonist beta-funaltrexamine. Milk engages the endogenous opioid system of the fetal rat and affects fetal responsiveness by interacting with the kappa receptors of the opioid system.

Behavior of rat fetuses following chemical or tactile stimulation.

The behavior of fetal rats was examined on Day 19 of gestation with procedures that enabled chemical stimulation and direct observation of fetuses. Rat fetuses are sensitive to both tactile stroking and intraoral infusion of chemical solutions, but the pattern and amount of activity depend upon the modality of stimulation. Fetal responsiveness is affected by prior experience with chemical stimuli. Repeated exposure within a 10-min period results in a waning of response, and repeated exposure across a delay of 48 hr results in a different pattern of response than is seen to a novel stimulus. Reexposure to a stimulus experienced earlier in gestation also alters fetal responsiveness to other forms of tactile and chemical stimulation. These findings indicate that the rat fetus exhibits olfactory function in utero and suggest central processing of sensory information, including evidence of habituation, a fetal orienting reflex to novel stimuli, and the existence of prenatal behavioral states associated with different patterns of response.

Stimulus contingencies that permit classical conditioning of opioid activity in the rat fetus.

In a prenatal model of classical conditioning, rat fetuses received presentations of an artificial nipple (conditioned stimulus; CS) paired with milk (unconditioned stimulus). Infusion of milk promotes activity in the kappa opioid system of the fetus, but after 2, 3, or 6 pairings with the artificial nipple, milk evoked both kappa and mu opioid activity. The nipple CS has no effect on opioid activity, but after pairing with milk evoked a mu opioid response. Conditioned mu opioid activity was evident in 60% of subjects tested after 1 paired conditioning trial. Significantly more fetal subjects (90%) exhibited conditioned opioid activity if preexposed to the nipple twice before conditioning. CS preexposure altered behavior during the conditioning trial, with preexposed fetuses showing more pronounced responses to milk infusion. Exposure to familiar stimuli facilitates classical conditioning of physiological responses, including opioid activity, during the first suckling episode.

Uterine position and conditioned taste aversion.

Three experiments were designed to determine the influence of uterine position on the performance of female rats in a conditioned taste aversion paradigm. The first and second experiments confirmed a differential behavioral response by males and females during acquisition and extinction of the conditioned taste aversion. However, no differences were found between females that had caudal male littermates in utero (MF) and females that had no caudal male littermates (FF). In the third experiment, in which testosterone was administered to females throughout testing, MF females showed an increased sensitivity to testosterone and a more prolonged rate of extinction than FF females. Exposure to testosterone during prenatal development heightened postnatal responsiveness to testosterone in female rats. The results are discussed in terms of the organizational and activational effects of testosterone on behavior in a conditioned taste aversion situation.

Cryptopsychobiology: the appearance, disappearance, and reappearance of a species-typical action pattern during early development.

Late in gestation, intraoral infusion of lemon elicits a facial wiping response from rat fetuses. This facial wiping response is isomorphic with that of older pups and adult rats exposed to aversive oral stimulation. Most studies of the postnatal development of aversive responses have demonstrated that facial wiping does not appear in the repertoire of rat pups until the second postnatal week. In certain test situations, however, wiping can be elicited from neonatal rats. This fact suggests that the expression of facial wiping by neonates is constrained or facilitated by the environmental conditions present at the time of testing. In this report, a series of seven experiments is described that document the wiping response of rat fetuses and pups in age-typical environments, and an environmental constraint hypothesis is examined. Examination of the ontogeny of facial wiping in this manner highlights issues that should be addressed in studies of behavioral continuity between the prenatal and the postnatal periods.

The rat fetus in its environment: behavioral adjustments to novel, familiar, aversive, and conditioned stimuli presented in utero.

With the pregnant rat under ether anesthesia, rat fetuses were exposed on Day 17 of gestation to a taste/odor stimulus (mint) injected into the amniotic fluid and/or lithium chloride injected into the peritoneum. Behavior of injected fetuses was directly observed on Day 19 of gestation following chemomyelotomy and laparotomy of the female and immersion of the uterus into a warm saline bath. With these procedures, a series of four experiments was conducted to assess the behavioral effects of (a) the mint taste/odor alone, (b) the LiCl alone, (c) the pairing of mint and LiCl on the day of conditioning, and (d) the reexposure to mint after an earlier pairing of mint and LiCl. These experiments provide clear evidence that rat fetuses are capable of forming conditioned taste/odor aversions as early as Day 17 of gestation and, further, that rat fetuses are capable of expressing these learned aversions in utero.

An intrinsic source of behavioral regulation that influences discrete responses to cues important for the initiation of suckling.

The present experiment investigated the relationship between motor activity and oral grasping of an artificial nipple in newborn rats. Pups orally grasped the artificial nipple, and they performed more and longer oral grasps in the latter portion of the nipple presentation. Motor activity was cyclical, and this cyclicity was evident before and during presentation of the artificial nipple. The onset of an oral grasp response was preceded by a period of relatively low motor activity, and the termination of a grasp was followed by relatively high motor activity. The newborn rat pup's intrinsic oscillations in motor activity may regulate the expression of discrete responses to cues important for the initiation of suckling.

Milk delivery schedules and stomach preloading alter patterns of suckling behavior by newborn rats on a surrogate nipple.

Newborn rat pups tested before suckling experience attached to and ingested milk from the surrogate nipple. Time attached to the nipple and amount of milk ingested depended on the schedule of milk infusion through the nipple. More frequent milk infusions resulted in more frequent disengagements from the nipple during the test, less time attached to the nipple, and less body weight gain. The initial patterns of attachment behavior--continuous or intermittent--were reproduced later when rats were tested on the surrogate nipple. Preloading of the stomach with milk effectively altered both attachment and ingestion from the nipple, whereas preloading with the same amount of water had no effect on suckling behavior. The data suggest that newborn rats flexibly adjust their attachment behavior to peculiarities of milk delivery through the surrogate nipple and reproduce the initial attachment pattern when reexposed to the surrogate nipple.

Cocaine alters behavior in the rat fetus.

Changes in motor behavior and sensory responsiveness were characterized in rat fetuses on gestational Day 21 after acute administration of various doses of cocaine. An increase in fetal motor activity was evident in the 3 highest doses (5, 10, and 20 mg/kg). Cocaine-exposed Ss showed reduced facial wiping in behavioral bioassays of cutaneous sensitivity (10 and 20 mg/kg) and chemosensory responsiveness (20 mg/kg). Changes in other behavioral measures indicated that fetuses detected and responded to these stimuli, suggesting that reduced facial wiping was due to a disruption of sensorimotor integration or motor coordination. Study of the fetus in vivo can provide insights into the mechanisms of cocaine's deleterious effects on central nervous system and behavioral development.

Central administration of cocaine produces age-dependent effects on behavior in the fetal rat.

Rat fetuses were exposed to cocaine, lidocaine, or saline on Gestational Day 20 or 21 to provide information about cocaine effects on behavior during prenatal development. Cocaine was administered into the cisterna magna of individual fetal subjects to restrict effects to the CNS. Behavioral effects of cocaine were compared with lidocaine to help distinguish the effects of cocaine on monoamine systems in the brain from its properties as a local anesthetic. Cocaine promoted 3-5-fold increases in fetal motor activity in the absence of explicit sensory stimulation, in contrast to the slight suppressive effects of lidocaine. Cocaine and lidocaine also reduced coordinated behavioral responses to an artificial nipple. The behavioral effects of cocaine administered into the CNS of fetal subjects suggest specific mechanisms of action on developing neural and behavioral systems in the late prenatal period.

Ontogeny of the stretch response in the rat fetus: kappa opioid involvement.

Previous studies have demonstrated that the kappa opioid system is functional and plays a role in mediating the stretch response of the rat fetus on Day 21 of gestation. In this study, a kappa opioid agonist (U50,488) was administered on Days 19, 20, or 21, and fetal behavior was recorded after infusion of either milk or saline. Activation of the kappa opioid system promoted stretching in response to saline on Days 20 and 21. Although fetuses on Day 19 did not stretch, videotape analysis indicated that kappa opioid manipulation promoted modest increases in rearlimb activity and changes in fetal body posture that typically occur antecedent to the stretch. These findings suggest that functional maturity, of the kappa opioid system may be a limiting factor in the expression of the fetal stretch response.

Endogenous AVP systems regulate oral behavior in the rat fetus: neuropeptide systems as ontogenetic adaptations.

Pharmacological manipulation of V1 receptors in rostral and caudal brain regions alters perioral responsiveness in the E20 rat fetus. Blockade of caudal V1 receptors or activation of rostral V1 receptors reduces fetal responsiveness to perioral cutaneous stimulation. Activation of caudal V1 receptors or blockade of rostral V1 receptors increases fetal responsiveness to perioral stimulation, including oral capture and grasping of an artificial nipple. These results suggest that V1 receptor-containing neurons regulate perioral responsiveness in the E20 rat fetus and that the 2 populations of neurons exhibit functional differences. The caudal part of the arginine8-vasopressin (AVP) system increases whereas the rostral part decreases responsiveness to different types of perioral stimuli. The neuropeptide AVP may affect suckling behavior immediately after birth by regulating perioral sensory responsiveness.

Fetal experience with milk or an artificial nipple alters appetitive and aversive responses to perioral cutaneous stimuli.

Fetal rats exhibit oral grasping of an artificial nipple. The authors examined interactive effects of sensory stimuli normally encountered in the suckling environment on subsequent responses to the nipple. Embryonic Day 20 rat fetuses received an infusion of milk, lemon, or saline through a hollow artificial nipple or an intraoral cannula (producing no nipple stimulation). One minute after sensory pretreatment, behavioral responses of fetuses to an artificial nipple were recorded on videotape for frame-by-frame analysis. Preexposure to the artificial nipple decreased the number of oral grasps and facial wipes directed toward the artificial nipple but increased the duration of grasp responses. Milk uniformly reduced fetal responsiveness to the nipple. Furthermore, the artificial nipple enhanced fetal responses to perioral cutaneous stimulation, whereas milk suppressed perioral responsiveness. These data suggest that the perinatal rat's 1st experience with milk or the nipple can alter subsequent responses to suckling stimuli.